Biofilms are associated with hundreds of thousands of infections each year and greatly increase treatment costs and mortality. These adverse effects are caused not only by increased resistance of bacteria living in the biofilm matrix but likely also by adaptive evolution of mutants into different, recalcitrant forms. To study this evolutionary process in biofilms we devised a method enabling long-term selection of populations of the opportunistic pathogen Burkholderia cenocepacia that undergo a cycle of attachment, biofilm assembly, and dispersal. Selection repeatedly favored mutations in loci commonly mutated during chronic infections, suggesting that biofilm adaptation recapitulates aspects of evolution during infections. This proposal focuses on two genetic loci that coordinate the sensing of a secreted signal (BDSF) with internal regulation of a switch governing biofilm production (cyclic-di-GMP). Mutations in different protein domains led to different ecological strategies and together different mutants produced a more robust biofilm. The overarching goal of this project is to precisely define the selective advantages of mutants with different strategie of signaling, adhesion, and dispersal in diverse biofilm environments. To tackle this complex problem we have assembled a multidisciplinary team with expertise in molecular genetics and evolutionary biology, biochemistry and mass spectrometry, and biophysical structure-function analysis. Our objectives are to 1) quantify how mutants that vary in sensing BDSF and producing cyclic-di-GMP are adaptive in biofilm communities, 2) determine how BDSF mechanistically controls the activity of the primary locus under selection, and the effects of adaptive mutations on this process, and 3) to develop an ecological model of how these processes operate in mixed-mutant and mixed-species communities. We expect to learn how this system may be manipulated to induce dispersal from biofilms and increase their susceptibility, which could be broadly relevant for developing novel antimicrobials.